In this chapter the characteristics and applications of Chemiluminescence (CL) as detection system coupled with Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC) and Capillary Electrophoresis (CE) are presented. Considering the advantages of CL related to its high sensitivity and selectivity or the simplicity of the required instrumentation (no external light source is needed) and the potential of GC, HPLC or CE as the most useful separation techniques with great efficiency for multianalyte determinations, the combination of those methodologies has permitted the development of a wide variety of powerful analytical methods. Progress in the different systems developed, commercial and home-made configurations and analytical applications in different fields, such as clinical, pharmaceutical, environmental and food analysis, have been discussed here.